I am interested in knowing the expression of a certain protein in the islets under feeding and fasting conditions. I use the same total protein concentration for immunolotting. In my first trial I used alpha tubulin as my internal control and found it gave me lighter band in the fasting condition. I thought it is a sampling error and I didn't adjust the same total protein concentration well but on repeating the experiment I got the same results. I tried reprobing the membranes with beta actin antibody for both of these experiments and I found it is less expressed in the islets as well on fasting.
Hi Dina
It may be that these two proteins (tubulin and beta actin) are down regulated in fasting conditions but there are other possible explanations for your result. Two things you may try are:
1.- weight the same amount of islets and determine protein to check similar protein amount per g of tissue.
2.- dialyze before protein determination to see whether small compounds or small peptides are "contaminating" and giving you an underestimation of proteins in fasting (or overestimation in non-fasting) conditions. For "dialysis" I would use a gel permeation PD-10 desalting column (the contain Sephadex G-25 resin). Please note that PD-10 will eliminate both small compounds and small peptides/proteins (your protein will be excluded or not depending on the molecular weight)
- Badges
- Science method
- Similar topics
- Biological Science
- Genetics
- Gene Expression
- Protein Expression